Kim Suwha, Choi Hyunwoo, Park Zee-Yong
Department of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea.
Mol Cells. 2007 Jun 30;23(3):340-8.
Although considerable effort has been devoted in the mass spectrometric analysis of phosphorylated peptides, successful identification of multi-phosphorylated peptides in enzymatically digested protein samples still remains challenging. The ionization behavior of multi-phosphorylated peptides appears to be somewhat different from that of mono- or di-phosphorylated peptides. In this study, we demonstrate increased sensitivity of detection of multi-phosphorylated peptides of beta casein without using phosphopeptide enrichment techniques. Proteinase K digestion alone increased the detection limit of beta casein multi-phosphorylated peptides in the LC-MS analysis almost 500 fold, compared to conventional trypsin digestion (~50 pmol). In order to understand this effect, various factors affecting the ionization of phosphopeptides were investigated. Unlike ionizations of phosphopeptides with minor modifications, those of multi-phosphorylated peptides appeared to be subject to effects such as selectively suppressed ionization by more ionizable peptides and decreased ionization efficiency by multi-phosphorylation. The enhanced detection limit of multi- phosphorylated peptides resulting from proteinase K digestion was validated using a complex protein sample, namely a lysate of HEK 293 cells. Compared to trypsin digestion, the numbers of phosphopeptides identified and modification sites per peptide were noticeably increased by proteinase K digestion. Non-specific proteases such as proteinase K and elastase have been used in the past to increase detection of phosphorylation sites but the effectiveness of proteinase K digestion for multi-phosphorylated peptides has not been reported.
尽管在磷酸化肽段的质谱分析方面已经投入了大量精力，但在酶解蛋白质样品中成功鉴定多磷酸化肽段仍然具有挑战性。多磷酸化肽段的电离行为似乎与单磷酸化或双磷酸化肽段有所不同。在本研究中，我们证明了在不使用磷酸肽富集技术的情况下，检测β-酪蛋白多磷酸化肽段的灵敏度有所提高。仅用蛋白酶K消化，与传统的胰蛋白酶消化（约50 pmol）相比，在液相色谱-质谱分析中β-酪蛋白多磷酸化肽段的检测限提高了近500倍。为了理解这种效应，研究了影响磷酸肽段电离的各种因素。与修饰较少的磷酸肽段的电离不同，多磷酸化肽段的电离似乎受到更多可电离肽段选择性抑制电离和多磷酸化导致电离效率降低等影响。使用复杂的蛋白质样品，即HEK 293细胞裂解物，验证了蛋白酶K消化导致多磷酸化肽段检测限提高的结果。与胰蛋白酶消化相比，蛋白酶K消化显著增加了鉴定出的磷酸肽段数量和每个肽段的修饰位点数量。过去曾使用蛋白酶K和弹性蛋白酶等非特异性蛋白酶来增加磷酸化位点的检测，但蛋白酶K消化对多磷酸化肽段的有效性尚未见报道。
